NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) is a family of transcription factors that consists of hetero- or homo-dimers of p65 (RelA), c-Rel, RelB, p50 (NF-κB1), and p52 (NF-κB2) (Dejardin 2006). In the classical, or canonical pathway of NF-κB, stimulation of a variety of cell membrane receptors leads to phosphorylation, ubiquitination, and proteasomal degradation of the IκBs (inhibitor proteins), which results in the nuclear translocation of the p65/50 hetero-dimer that turns on transcription. The canonical path can be effectively blocked by inhibition of IκBβ kinase, 26S proteasome, or p65 binding to DNA. The alternative, or non-canonical path, is regulated through proteolysis of the inhibitory ankyrin containing protein NF-κB2/p100 to release p52, which typically dimerizes with RelB. In addition, there is a “hybrid” path that activates p52/c-Rel and p52/p65. The non-canonical or “hybrid” paths are not susceptible to IκBβ kinase or proteasomal inhibitors. These paths are most effectively inhibited by antagonizing RelB or c-Rel binding to DNA. The canonical and non-canonical paths are associated with different aspects of specific diseases through activations of distinctive groups of genes. Thus, selective inhibition of either the canonical, or noncanonical pathway, or both, under different disease states, is believed to be a most effective approach to ameliorate the underlining disease conditions.
NF-κB activation has been implicated in a wide variety of diseases, including cancer, AIDS, diabetes mellitus, cardiovascular diseases, autoimmune diseases, viral replication, septic shock, neurodegenerative disorders, ataxia telangiectasia (AT), arthritis, asthma, inflammatory bowel disease, and other inflammatory conditions, atherosclerosis, heart disease, asthma, catabolic disorders, type 1 and 2 diabetes, ageing, skin diseases, renal diseases, gut diseases, pancreatitis, neuropathological diseases, pulmonary diseases, chronic obstructive pulmonary disease, sepsis and sleep apnea. The activation of NF-κB has been implicated in a large number of human diseases.
For example, activation of NF-κB by Gram-negative bacterial lipopolysaccharides (LPS) may contribute to the development of septic shock because NF-κB over-activates the transcription of numerous cytokines and modifying enzymes, whose prolonged expression can negatively effect the function of vital organs such as the heart and liver (Arcaroli et al., 2006; Niu et al., 2008).
Additionally, in chronic Alzheimer's disease, the amyloid β peptide causes production of reactive oxygen intermediates and indirectly activates gene expression through NF-κB sites (Giri et al., 2005).
Destructive erosion of bone or osteolysis is a major complication of inflammatory conditions such as rheumatoid arthritis (RA), periodontal disease, and periprosthetic osteolysis. RA is an autoimmune disease that affects approximately 1.0% of US adults, with a female to male ratio of 2.5 to 1 (Lawrence et al., 1998). Its hallmark is progressive joint destruction which causes major morbidity. Periodontal disease is highly prevalent and can affect up to 90% of the world's population. It is well known as the leading cause of tooth loss in adults (Pihlstrom et al., 2005). Despite its prevalence, little is known about the mechanism by which periodontal bone erosion occurs, although host response to pathogenic microorganisms present in the mouth appears to trigger the process. Periprosthetic osteolysis is caused by chronic bone resorption around exogenous implant devices until fixation is lost (Harris, 1995), and is considered as resulting from an innate immune response to wear-debris particles, with little contribution by components of the acquired immune system (Goldring et al., 1986).
Although these conditions are initiated by distinct causes and progress by alternative pathways, the important common factor(s) in the pathological process of these diseases are over-production of proinflammatory cytokines which is driven by the constitutive activation of the NF-κB pathway in the inflamed tissue. The bone erosion seen in these conditions is largely localized to the inflamed tissues, distinct from systemic, hormonally regulated bone pathologies, such as osteoporosis. These inflamed tissues, found in many of these diseases, also produce proinflammatory cytokines, i.e., TNF-α, IL-1, and IL-6, that are, in turn, involved in osteoclast differentiation signaling and bone-resorbing activities. Thus, inflammatory osteolysis is the product of enhanced osteoclast recruitment and activation prompted by NF-κB driven proinflammatory cytokines in the inflamed tissue.
Inflammatory bowel disease (IBD) encompasses a number of chronic relapsing inflammatory disorders involving the gastrointestinal tract. The two most prevalent forms of IBD, Crohn's disease and ulcerative colitis, can be distinguished by unique histopathologies and immune responses (Atreya et al., 2008; Bouma & Strober, 2003). The limited efficacy and potential adverse effects of current treatments leave patients and doctors eager for new treatments to manage the chronic relapsing inflammatory nature of these diseases.
Although the exact aetiologies leading to Crohn's disease and ulcerative colitis remain unknown, they are generally thought to result from an inappropriate and ongoing activation of the mucosal immune system against the normal luminal flora (Tilg et al., 2008). As a result, resident macrophages, dendritic cells and T cells are activated and begin to secrete predominantly NF-κB-dependent chemokines and cytokines. NF-κB mediated overproduction of key pro-inflammatory mediators is attributed to the initiation and progression of both human IBD and animal models of colitis (Neurath et al., 1998; Wirtz & Neurath, 2007). In particular, macrophages of patients with IBD exhibit high levels of NF-κB DNA binding activity accompanied by increased production of interleukin (IL)1, IL6 and tumour necrosis factor (TNFα) (Neurath et al., 1998). In addition, NF-κB plays a vital role in activating T helper cell 1 (Th1) and T helper cell 2 (Th2) cytokines, both of which are required for promoting and maintaining inflammation (Barnes, 1997). Because of the central role played by NF-κB in IBD, extensive efforts have been made to develop treatments targeting this pathway.
NF-κB has been shown to be constitutively expressed in numerous cancer derived cell lines from breast, ovarian, colon, pancreatic, thyroid, prostate, lung, head and neck, bladder, and skin tumors (Calzado et al., 2007). This has also been seen for B-cell lymphoma, Hodgkin's disease, T-cell lymphoma, adult T-cell leukemia, acute lymphoblastic leukemia, multiple myeloma, chronic lymphocytic leukemia, and acute myelogenous leukemia. Although NF-κB is a key mediator of normal inflammation as part of the defense response, chronic inflammation can lead to cancer, diabetes, and a host of other diseases as mentioned above. Several pro-inflammatory gene products have been identified that mediate a critical role in the carcinogenic process, angiogenesis, invasion, and metastasis of tumor cells. Among these gene products are TNF and members of its superfamily, IL-1α, IL-1β, IL-6. IL-8, IL-18, chemokines, MMP-9, VEGF, COX-2, and 5-LOX. The expression of all these genes are mainly regulated by the transcription factor NF-κB, which is constitutively active in most tumors and is induced by carcinogens (such as cigarette smoke), tumor promoters, carcinogenic viral proteins (HIV-tat, KHSV, EBV-LMP1, HTLV1-tax, HPV, HCV, and HBV), chemotherapeutic agents, and gamma-irradiation (Aggarwal et al., 2006). These observations imply that anti-inflammatory agents that suppress NF-κB should have a potential in both the prevention and treatment of cancer.
The influenza virus protein hemagglutinin also activates NF-κB, and this activation may contribute to viral induction of cytokines and to some of the symptoms associated with influenza (Flory et al., 2000; Pahl & Baeuerle, 1995).
Oxidized lipids from the low density lipoproteins associated with atherosclerosis activate NF-κB, which then activates other genes such as inflammatory cytokines (Liao et al., 1994). Furthermore, mice that are susceptible to atherosclerosis, exhibit NF-κB activation when fed an atherogenic diet, due to their susceptibility to aortic atherosclerotic lesion formation associated with the accumulation of lipid peroxidation products, induction of inflammatory genes, and the activation of NF-κB transcription factors (Liao et al., 1994). Another important contributor to atherosclerosis is thrombin, which stimulates the proliferation of vascular smooth muscle cells through the activation of NF-κB (Maruyama et al., 1997). A truncated form of IκB repressor protein (IκBα) was shown to be the cause of the hypersensitivity to ionizing radiation and is defective in the regulation of DNA synthesis in ataxia telangiectasia (AT) cells, which have constitutive levels of NF-κB-activation (Jung et al., 1995). This mutation in IκBα from the AT cells was shown to inactivate the repressor protein causing the constitutive activation of the NF-κB pathway. In light of all these findings, the abnormal activation or expression of NF-κB is clearly associated with a wide variety of pathologic conditions.
The infection and life-cycle of HIV-1 is tightly coupled to the NF-κB pathway in human mononuclear cells. Viral infection leads to the activation of NF-κB which generates the over stimulation and eventual depletion of T-cells that is the hallmark of AIDS (reviewed in (Argyropoulos & Mouzaki, 2006). For instance, the expression of CCR5, a key receptor for HIV-1, is regulated by NF-κB (Liu et al., 1998). Deletion analysis of the CCR-5 promoter has demonstrated that loss of the 3′-distal NF-κB/AP-1 site drops transcription by >95% (Liu et al., 1998). These studies would suggest that constitutive repression of NF-κB would cause a dramatic decrease in CCR-5 receptor message. Since HIV-1 entry kinetics are influenced by expressed levels of CCR5 on the target T-cell surface (Ketas et al., 2007; Platt et al., 1998; Reeves et al., 2002), down modulating CCR5 may constrain the expansion of the pool of infected cells that spawns the viral reservoir. CXCR4 expression has also been reported to be influenced by NF-κB (Helbig et al., 2003) suggesting that NF-κB inhibitors may be equally effective against X4-tropic isolates that appear during late-stage infection. NF-κB is required for transcription of the integrated DNA-pro-virus (Baba, 2006; Iordanskiy et al., 2002; Mukerjee et al., 2006; Palmieri et al., 2004; Rizzi et al., 2004; Sui et al., 2006; Williams et al., 2007). In fact, lack of NF-κB activation leads to the generation of a population of cells harboring latent virus which is a major block to eliminating the virus from infected patients (Williams et al., 2006).
NF-κB promotes the expression of over 150 target genes in response to inflammatory stimulators. These genes include interleukin-1, -2, -6 and the tumor necrosis factor receptor (TNF-R) (these receptors mediate apoptosis, and function as regulators of inflammation), as well as genes encoding immunoreceptors, cell adhesion molecules, and enzymes such as cyclooxygenase-II and inducible nitric oxide synthase (iNOS) (Karin, 2006; Tergaonkar, 2006). It also plays a key role in the progression of diseases associated with viral infections such as HCV and HIV-1.
Members of the NF-κB family include RelA/p65, RelB, c-Rel, p50/p105 (NF-κB)), and p52/p100 (NF-κB2) (Hayden & Ghosh, 2004; Hayden et al., 2006a; Hayden et al., 2006b). The Rel family members function as either homodimers or heterodimers with distinct specificity for cis-binding elements located within the promoter domains of NF-κB-regulated genes (Bosisio et al., 2006; Natoli et al., 2005; Saccani et al. 2004). Classical NF-κB, composed of the RelA p65 and p50 heterodimer, is the best-studied form of NF-κB (Burstein & Duckett, 2003; Hayden & Ghosh, 2004) and references therein). Prior to cellular stimulation, classical NF-κB resides in the cytoplasm as an inactive complex bound to the IκBα inhibitor proteins. Inducers of NF-κB such as bacterial lipopolysaccharides, inflammatory cytokines, or HIV-1 Vpr protein release active NF-κB from the cytoplasmic complex by activating the IκB-kinase complex (IKK), which phosphorylates IκBα (Greten & Karin, 2004; Hacker & Karin, 2006; Israel, 2000; Karin, 1999; Scheidereit, 2006). Phosphorylation of IκB marks it for subsequent ubiquitinylation and degradation by the 26S proteosome. Free NF-κB dimers translocate into the nucleus where they stimulate the transcription of their target genes.
The molecular design of racemic dehydroxymethylepoxyquinomicin (DHMEQ) was based on the antibiotic epoxyquinomicin C isolated from Amycolatopsis (Chaicharoenpong et al. 2002). DHMEQ was synthesized as a racemate from 2,5-dimethoxyaniline in five steps. Separation of the enantiomers on a chiral column produced both (+) and (−) enantiomers. The (−)-enantiomer was shown to be more potent at inhibiting NF-κB than the (+)-enantiomer (Umezawa et al. 2004). DHMEQ has been shown to specifically inhibit the translocation of NF-κB into the nucleus (Ariga et al. 2002). Specifically, it covalently modifies a specific cysteine residue in p65 and other Rel homology proteins with a 1:1 stoichiometry ratio (Yammamoto et al. 2008). As an NF-κB inhibitor, DHMEQ has been tested extensively in various animal models of diseases and has demonstrated a broad spectrum of efficacy including treating solid tumors, hematological malignancy, arthritis, bowel ischemia, and atherosclerosis (Watanabe et al. 2006). Thus, DHMEQ may be useful as a treatment for cancer and inflammation (Takeuchi et al. 2003).

In view of the role that activation of NF-κB plays in a number of diverse diseases, such as those described above, there is an ongoing need for effective small-molecule NF-κB inhibitors.
Several series of small molecule inhibitors have been discovered that directly inhibit the binding of NF-κB components, p65 (RelA), RelB and c-Rel to DNA. As a result, these compounds can block both the canonical and the noncanonical paths of NF-κB. The dual inhibition is distinct from IκBβ kinase inhibitors that affect only the canonical path. The efficacies of the compounds have been tested in animal models of multiple myeloma and rheumatoid arthritis and the results are described herein.